TY - JOUR
T1 - Influence of DOTA chelator position on biodistribution and targeting properties of 111In-labeled synthetic anti-HER2 affibody molecules
AU - Perols, Anna
AU - Honarvar, Hadis
AU - Strand, Joanna
AU - Selvaraju, Ramkumar
AU - Orlova, Anna
AU - Eriksson Karlström, Amelie
AU - Tolmachev, Vladimir
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 2012/8/15
Y1 - 2012/8/15
N2 - Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with 99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule ZHER2:S1 at three different positions: DOTA-A1-ZHER2:S1 (N-terminus), DOTA-K58-ZHER2:S1 (C-terminus), and DOTA-K50-ZHER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the KD values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-ZHER2:S1, DOTA-K50-ZHER2:S1, and DOTA-K58-Z HER2:S1, respectively. ZHER2:S1-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-ZHER2:S1 (64 °C) and DOTA-K58-ZHER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with 111In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-ZHER2:S1and DOTA-K58-ZHER2:S1 showed the highest uptake. Overall, DOTA-K58-Z HER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.
AB - Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with 99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule ZHER2:S1 at three different positions: DOTA-A1-ZHER2:S1 (N-terminus), DOTA-K58-ZHER2:S1 (C-terminus), and DOTA-K50-ZHER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the KD values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-ZHER2:S1, DOTA-K50-ZHER2:S1, and DOTA-K58-Z HER2:S1, respectively. ZHER2:S1-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-ZHER2:S1 (64 °C) and DOTA-K58-ZHER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with 111In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-ZHER2:S1and DOTA-K58-ZHER2:S1 showed the highest uptake. Overall, DOTA-K58-Z HER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.
UR - http://www.scopus.com/inward/record.url?scp=84865145661&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84865145661&partnerID=8YFLogxK
U2 - 10.1021/bc3002369
DO - 10.1021/bc3002369
M3 - Article
C2 - 22768790
AN - SCOPUS:84865145661
VL - 23
SP - 1661
EP - 1670
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
SN - 1043-1802
IS - 8
ER -